Hepoxilin A3 Facilitates Neutrophilic Breach of Lipoxygenase-Expressing Airway Epithelial Barriers

A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A3 is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A3 in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A3, including phospholipase A2 and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A3 precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A3 are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A3 and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A3 serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A3 are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A3 and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A3 is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.